Maternal auxin signaling inhibits seed germination in Arabidopsis, but little is known about the gene network that mediates the inhibition of seed germination by auxin. Based on publicly available data, we first determined the expression patterns of AUXIN SIGNALING F-BOX (AFB) genes in the funiculus (FUN)-chalazal seed coat (CSC) continuum. We found that TIR1, AFB1, and AFB4 exhibited a down-expression gradient and AFB2, AFB3, and AFB5 an up-expression gradient from FUN to CSC, and members in each of the two AFB groups were linearly correlated in expression in FUN, CSC, and the distal seed coat (DSC). We then searched for auxin-regulated genes that exhibited one of the two expression gradients. We found 118 such genes that were assigned into four groups based on their response mode to auxin and expression gradient direction. The four groups were further broken down into 12 subgroups based on the linear correlation coefficient values. Only three of the 12 subgroups, including a total of 30 genes with 21 of them being known or highly likely to function in the seed germination process. To explore whether additional genes of the remaining nine play roles in seed germination, we tested mutants of five of them in a germination assay and found all of them exhibited either delayed or hastened germination. The experimental results support the validity of our approach for predicting the involvement of these genes in seed germination. Based on publicly available data and data from this investigation, we constructed a gene network, which should provide a valuable framework and new clues for future studies of the molecular mechanism controlling seed germination.

Polyploidization promotes the functional diversification of autophagy in plants, expanding autophagy-associated genes (AAGs) to support processes like chloroplast division and flowering. Analysis of 92,967 AAGs in Arabidopsis thaliana, Solanum lycopersicum, Camellia oleifera, and 74 other plant species shows that 45.69% of AAGs are polyploidy-related, highlighting polyploidy's role in linking autophagy to plant-specific functions.

Evolution of cellular characteristics is a fundamental aspect of evolutionary biology, but knowledge about evolution at the cellular level is very limited. In particular, whether a certain intracellular characteristic evolved in angiosperms, and what significance of such evolution is to angiosperms, if it exists, are important and yet unanswered questions. We have found that bidirectional cytokinesis occurs or likely occurs in male meiosis in extant basal and near-basal angiosperm lineages, which differs from the unidirectional cytokinesis in male meiosis in monocots and eudicots. This pattern of cytokinesis in angiosperms seems to align with the distribution pattern of angiosperms with the lineages basal to monocots and eudicots living in tropical, subtropical or temperate environments and monocots and eudicots in an expanded range of environments including tropical, subtropical, temperate, subarctic and arctic environments. These two cytokinetic modes seem to result from two phragmoplast types, respectively. A phragmoplast in the bidirectional cytokinesis dynamically associates with the leading edge of a growing cell plate whereas a phragmoplast in the unidirectional cytokinesis is localized to an entire division plane. The large assembly of microtubules in the phragmoplast in unidirectional cytokinesis may be indicative of increased microtubule stability compared with that of the small microtubule assembly in the phragmoplast in bidirectional cytokinesis. Microtubules could conceivably increase their stability from evolutionary changes in tubulins and/or microtubule-associated proteins. Microtubules are very sensitive to low temperatures, which should be a reason for plants to be sensitive to low temperatures. If monocots and eudicots have more stable microtubules than other angiosperms, they will be expected to deal with low temperatures better than other angiosperms. Future investigations into the male meiotic cytokinetic directions, microtubule stability at low temperatures, and proteins affecting microtubule stability in more species may shed light on how plants evolved to inhabit cold environments.

Abstract The plant hormone auxin suppresses seed germination, but how auxin does it remains poorly understood. While studying the functions of the AUXIN SIGNALING F-BOX (AFB) auxin co-receptors in Arabidopsis, we consistently isolated AFB1 and AFB5 in reproductive tissues in co-immunoprecipitation experiments using their interacting protein ASK1 as the bait. However, T2 seeds of the AFB1 or AFB5 transgenic lines generated for the co-immunoprecipitation experiments frequently failed to germinate, which led to the studies of seed germination in these plants and afb1 and afb5 mutants, and AFB1 and AFB5 expression in nearly mature fruit and imbibed seeds using AFB1:GUS and AFB5:GUS lines. We found that AFB1 and AFB5 acted in maternal tissues to suppress seed germination and their effects were positively correlated with the plants’ sensitivity to indole acetic acid. Conversely, afb1 and afb5 single mutants exhibited faster seed germination than the wild type and the seeds of the afb1-5afb5-5 double mutant germinated even faster than those of the afb1-5 and afb5-5 single mutants. Seed germination of the afb1-5afb5-5 double mutant also exhibited higher sensitivity to gibberellic acid than that of the wild-type and the afb1-3, afb1-5 and afb5-5 single mutants. Both AFB1 and AFB5 were expressed in the funiculus during seed maturation, and AFB1 was also transiently expressed in a small chalazal region surrounding the hilum in the seed coat during seed imbibition. Therefore, AFB1 and AFB5 likely suppress seed germination in the funiculus and AFB1 also briefly suppresses seed germination in the chalaza during seed imbibition.

Background:Complex haploinsufficiency is characterized by individuals with two heterozygous loci producing a phenotype that is not seen in either of the corresponding single-locus heterozygous individuals. The mutants of the anaphase-promoting complex/cyclosome (APC/C) subunit geneAPC11and the mitotic cyclin geneCYCLIN A1;2/TARDY ASYNCHRONOUS MEIOSIS(TAM) inArabidopsis thalianaare embryo-lethal and defective in meiosis, respectively, but their heterozygous single mutants do not exhibit defective embryo development and seed germination.Methods:Crosses between two heterozygousapc11mutant alleles and two homozygoustammutant alleles, and between two heterozygousapc11mutant alleles and aTAM:TAM-GFPline were conducted. Phenotypes of the F1seeds were analyzed by light microscopy.Results:We found that F1embryos from the crosses between heterozygousapc11-1(APC11/apc11-1) and homozygoustam-2(tam-2/tam-2) or betweenAPC11/apc11-2andtam-2/tam-2were morphologically normal but all the seeds failed to germinate. F1embryos from the crosses betweenAPC11/apc11-2andtam-1/tam-1(weaker allele thantam-2) produced morphologically normal seeds that germinated to form mature plants. However, F1embryos from the crosses betweenAPC11/apc11-1andtam-1/tam-1were abnormal and the seeds failed to germinate. Moreover, F1embryos from the crosses betweenAPC11/apc11-1and aTAM:TAM-GFPline were arrested at early developmental stages while F1embryos from the crosses betweenAPC11/apc11-2and theTAM:TAM-GFPline appeared fully developed but the seeds failed to germinate.Conclusions:Our observations indicate that theapc11andtammutants have an allele-dependent complex haploinsufficient relationship in embryo development and seed germination.

Comparing cellular features in microsporogenesis across taxa may yield important clues to evolution of meiosis in plants. We previously provided evidence that bidirectional cytokinesis occurs in M. denudata and suggested that the same may also occur in P. trimera based on a published report. Both M. denudata and P. trimera are basal angiosperm species that belong to the order of Magnoliales. For comparison, only unidirectional cytokinesis, either centripetal or centrifugal cytokinesis, has been found in microsporogenesis in eudicots and monocots. These observations raise the possibility that bidirectional cytokinesis is a common feature of microsporogenesis in basal angiosperms but not in eudicots and monocots. In this report, we provide evidence that bidirectional cytokinesis also occurs in another basal angiosperm species, Nymphaea colorata. The new findings, together with the previous findings, indicate that bidirectional cytokinesis is a prominent feature of microsporogenesis in at least some basal angiosperm species, and it occurs independently of cytokinesis types with respect to the timing of cytokinesis and tetrad configurations.

Magnolia, a basal angiosperm genus important for evolutionary and phylogenetic studies, is known to have male meiotic features not seen in the vast majority of angiosperms. However, knowledge about male meiosis in Magnolia is still fragmentary. Here, we report findings from an extensive investigation into male meiosis in Magnolia denudata using a combination of light and electron microscopy methods. Male meiosis in M. denudata was synchronous in prophase I but asynchronous in subsequent nuclear divisions. The polarized microspore mother cells from late prophase I onward had an elongated cell shape and thickened callose wall areas at the two smaller ends of the cell. The first nuclear division occurred along the long axis of the cell and the first callose furrow formed at the equatorial plane of the first nuclear division at the late telophase I stage. The second equatorial callose furrow formed after telophase II in a plane perpendicular to the first callose furrow. While cytokinesis occurred centripetally from the two furrows, a central callose wall island (CWI) appeared in the center of the cell and dense assemblies of vesicles and short tubules decorated the cytoplasmic regions between the furrows and the CWI. This cytokinesis mode differs from either the centripetal or the centrifugal mode of cytokinesis in microsporogenesis in the vast majority of angiosperms. As a result of this unusual cytokinesis, a large central callose mass remains in the mature tetrads. These observations may be useful to studies of cytokinetic mechanisms, evolution of microsporogenesis, and phylogenetics of angiosperms.

Sustained oscillations are frequently observed in biological systems consisting of a negative feedback loop, but a mathematical model with two ordinary differential equations (ODE) that has a negative feedback loop structure fails to produce sustained oscillations. Only when a time delay is introduced into the system by expanding to a three-ODE model, transforming to a two-delay differential equations (DDE) model, or introducing a bistable trigger do stable oscillations present themselves. In this study, we propose another mechanism for producing sustained oscillations based on periodic reaction pauses of chemical reactions in a negative feedback system. We model the oscillatory system behavior by allowing the coefficients in the two-ODE model to be periodic functions of time-called pulsate functions-to account for reactions with go-stop pulses. We find that replacing coefficients in the two-ODE system with pulsate functions with microscale (several seconds) pauses can produce stable system-wide oscillations that have periods of approximately 1 to several hours long. We also compare our two-ODE and three-ODE models with the two-DDE, three-ODE, and three-DDE models without the pulsate functions. Our numerical experiments suggest that sustained long oscillations in biological systems with a negative feedback loop may be an intrinsic property arising from the slow diffusion-based pulsate behavior of biochemical reactions.

Cellulases, hemicellulases, and pectinases play important roles in fruit development and maturation. Although mutants with defects in these processes have not been reported for cellulase or hemicellulase genes, the pectinases ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1) and ADPG2 were previously shown to be essential for silique dehiscence in Arabidopsis (Arabidopsis thaliana). Here, we demonstrate that the cellulase gene CELLULASE6 (CEL6) and the hemicellulase gene MANNANASE7 (MAN7) function in the development and dehiscence of Arabidopsis siliques. We found that these genes were expressed in both vegetative and reproductive organs and that their expression in the silique partially depended on the INDEHISCENT and ALCATRAZ transcription factors. Cell differentiation was delayed in the dehiscence zone of cel6 and man7 mutant siliques at early flower development stage 17, and a comparison of the spatio-temporal patterns of CEL6 and MAN7 expression with the locations of delayed cell differentiation in the cel6 and man7 mutants revealed that CEL6 and MAN7 likely indirectly affect the timing of cell differentiation in the silique valve at this stage. CEL6 and MAN7 were also found to promote cell degeneration in the separation layer in nearly mature siliques, as cells in this layer remained intact in the cel6 and man7 mutants and the cel6-1 man7-3 double mutant, whereas they degenerated in the wild-type control. Phenotypic studies of single, double, triple, and quadruple mutants revealed that higher-order mutant combinations of cel6-1, man7-3, and adpg1-1 and adpg2-1 produced more severe silique indehiscent phenotypes than the corresponding lower-order mutant combinations, except for some combinations involving cel6-1, man7-3, and adpg2-1 Our results demonstrate that the ability of the silique to dehisce can be manipulated to different degrees by altering the activities of various cell wall-modifying enzymes.

Arabidopsis CEL6 and MAN7 proteins affect cell morphology and silique dehiscence, which can be manipulated to different degrees by altering their activities. Cellulases, hemicellulases, and pectinases play important roles in fruit development and maturation. Although mutants with defects in these processes have not been reported for cellulase or hemicellulase genes, the pectinases ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1) and ADPG2 were previously shown to be essential for silique dehiscence in Arabidopsis (Arabidopsis thaliana). Here, we demonstrate that the cellulase gene CELLULASE6 (CEL6) and the hemicellulase gene MANNANASE7 (MAN7) function in the development and dehiscence of Arabidopsis siliques. We found that these genes were expressed in both vegetative and reproductive organs and that their expression in the silique partially depended on the INDEHISCENT and ALCATRAZ transcription factors. Cell differentiation was delayed in the dehiscence zone of cel6 and man7 mutant siliques at early flower development stage 17, and a comparison of the spatio-temporal patterns of CEL6 and MAN7 expression with the locations of delayed cell differentiation in the cel6 and man7 mutants revealed that CEL6 and MAN7 likely indirectly affect the timing of cell differentiation in the silique valve at this stage. CEL6 and MAN7 were also found to promote cell degeneration in the separation layer in nearly mature siliques, as cells in this layer remained intact in the cel6 and man7 mutants and the cel6-1 man7-3 double mutant, whereas they degenerated in the wild-type control. Phenotypic studies of single, double, triple, and quadruple mutants revealed that higher-order mutant combinations of cel6-1, man7-3, and adpg1-1 and adpg2-1 produced more severe silique indehiscent phenotypes than the corresponding lower-order mutant combinations, except for some combinations involving cel6-1, man7-3, and adpg2-1. Our results demonstrate that the ability of the silique to dehisce can be manipulated to different degrees by altering the activities of various cell wall-modifying enzymes.

What values of relative numerical tolerance should be chosen in simulation of a deterministic model of a biochemical reaction is unclear, which impairs the modeling effort since the simulation outcomes of a model may depend on the relative numerical tolerance values. In an attempt to provide a guideline to selecting appropriate numerical tolerance values in simulation of in vivo biochemical reactions, reasonable numerical tolerance values were estimated based on the uncertainty principle and assumptions of related cellular parameters. The calculations indicate that relative numerical tolerance values can be reasonably set at or around 10−4 for the concentrations expressed in ng/L. This work also suggests that further reducing relative numerical values may result in erroneous simulation results.

New microarray chip strategies that are sensitive and selective and that can measure low levels of important biomarkers directly in a blood sample are significant for improving human health by allowing timely diagnosis of an abnormal condition. Herein, we designed an antibody-aptamer immunoarray chip to demonstrate simultaneous measurement of blood insulin and glycated hemoglobin (HbA1c) levels relevant to diabetic and prediabetic disorders using a surface plasmon microarray with validation by fluorescence imaging. To accomplish both surface plasmon and fluorescence imaging on the same sample, we decorated magnetite nanoparticles with quantum dots for covalent immobilization of aptamers for subsequent capture and isolation of the aptamers specific for insulin and HbA1c markers from 20-times diluted whole blood samples. Direct clinically relevant analysis, along with fluorescent imaging of the two markers, was achieved by this new immunoarray platform. The limit of detection was 4 pM for insulin and 1% for HbA1c. Examination of cross-talk using thrombin and platelet-derived growth factor confirmed that the designed immunoarray was highly selective for insulin and HbA1c. Surface plasmon kinetic analysis provided apparent binding constants of 0.24 (±0.08) nM and 37 (±3) μM, respectively, for the binding of insulin and HbA1c onto their surface immobilized monoclonal antibodies. Thus, quantitative imaging of ultralow levels of blood biomarker levels with binding kinetics is uniquely obtained in the designed immunoarray chip. In conclusion, this report demonstrates considerable significance of the developed magnetite-quantum dot-bioconjugate strategy for clinical diagnostics of whole blood biomarkers with characterization of molecular binding interactions.

In order to gain a better understanding of the function of SKIP16, a gene encoding the F-box protein of the SKP1-CULLIN-F-BOX PROTEIN (SCF) complex, on the reproductive development of Arabidopsis thaliana, we conducted an experiment with transgenic plants containing the gene ASK1:SKIP16. We did this to see the effect of overexpression of SKIP16 on the plants to further begin to understand the role of SKIP16 on Arabidopsis development since the ASK1 promoter ubiquitously drives high expression levels of SKIP16. While it is known that SCF complexes impact growth and development in plants, the function of SKIP16 is unknown. We observed that at the T1 generation, two of the ASK1:SKIP16 plants had only one terminal flower on each inflorescence stem while many other plants produced either infertile siliques or siliques with reduced fertility. These observations indicate that SKIP16 plays an important role in Arabidopsis reproductive development. Further research on the function of SKIP16 may yield crucial information about how SCF F-box protein regulates plant development in Arabidopsis and other plants.

PREMISE OF THE STUDY:The Arabidopsis MYB transcriptional factors FOUR LIPS (FLP) and MYB88 are predicted to play a broader role than just limiting the number of guard cells per stoma in the epidermis based on their expression patterns. Analysis of the numbers of all epidermal cells in cotyledons in flp mutants and the flp-1 myb88 double mutant may substantiate the functions of FLP and MYB88 in nonstomatal epidermal cells.METHODS:The number of all types of abaxial epidermal cells in mature cotyledons were determined under different growth conditions in flp-1, flp-3, flp-8, and tmm-1 single mutants, flp-1 tmm-1 and flp-1 myb88 double mutants, and accessions Columbia-0 (Col, control for flp-1, flp-3, and flp-1 myb88) and Landsberg erecta (Ler, control for flp-8).KEY RESULTS:In soil-grown plants, the number of pavement cells and meristemoids per cotyledon were not statistically different between the flp mutants and their respective controls and between flp-1 and flp-1 myb88, except sometimes for the number of meristemoids or pavement cells between flp-1 and Col. In contrast, the same comparisons yielded statistically significant differences in medium-grown plants, i.e., more cells in the flp mutants and flp-1 myb88, except for meristemoids between flp-8 and Ler and pavement cells between flp-1 and flp-1 myb88. No significant difference was detected for nonstomatal epidermal cells between tmm-1 and flp-1/tmm-1 under the two conditions.CONCLUSIONS:FLP and MYB88 inhibit the production of nonstomatal epidermal cells largely in a growth-condition-dependent manner. The tmm-1 mutation is epistatic to the flp-1 mutation in the production of nonstomatal epidermal cells in the cotyledon.

It is known that the tardy asynchronous meiosis-2 (tam-2) mutant of Arabidopsis thaliana produces unreduced gametes by skipping meiosis II, resulting in genome duplication in the next generation. This process of polyploidization continues until it reaches octoploidy that becomes unstable. The progeny of octoploid tam-2 are of reduced ploidy as a result. The purpose of this study was to examine fertility and morphological changes over at least two generations from the octoploid tam-2 of A. thaliana. We hypothesize that the instability of the genome may result in new genome compositions that produce new phenotypes in the generations after the octoploid generation. We found a positive linear relationship between guard cell nuclear area and ploidy level; this relation will be used for determining the ploidy levels in the progeny of the octoploid tam-2. By observing morphological changes due to genomic decay in real time, we may find new varieties of Arabidopsis in the making over one or more generations, which has important implications to the mechanisms of speciation, and to technological development for engineering new crops in the agricultural industry.

UNLABELLED:•PREMISE OF THE STUDY:Spatiotemporal features of microsporogenesis may provide important clues about the evolution of microsporogenesis in seed plants. One cellular feature that attracts special attention is advance cell wall ingrowths (ACWIs) at future cytokinetic sites in microsporocytes since they have been found only in species of an ancient lineage of angiosperms, Magnolia, and in much less detail, of an ancient lineage of gymnosperms, cycads. Further investigation into microsporogenesis in a cycad species may yield knowledge critical to understanding the establishment of ACWIs as an important feature for comparative studies of microsporogenesis in seed plants.•METHODS:Bright-field and epifluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy were used to investigate the microsporogenic process in Macrozamia communis, a species in the Zamiaceae family of cycads.•KEY RESULTS:In prophase-II microsporocytes in M. communis, ACWIs form as a callose ring between the newly formed nuclei and are not accompanied by cytokinetic apparatuses such as mini-phragmoplasts, wide tubules, or wide tubular networks. Shortly after the second nuclear division, new ACWIs, albeit thinner than the previous ACWIs, form between the newly formed nuclei. Subsequent cell plate formation in the planes of the ACWIs typically results in tetragonal tetrads.•CONCLUSIONS:Cytokinesis at the cell periphery is initiated earlier than cell plate formation in the cell interior in microsporogenesis in M. communis. The cellular features uncovered in M. communis may serve as useful reference features for comparative studies of microsporogenesis in plants.

In vitro microtubule assembly exhibits a rhythmic phenomenon, that is, fast growth periods alternating with slow growth periods. Mechanism underlying this phenomenon is unknown. Here a simple diffusion mechanism coupled with small diffusion coefficients is proposed to underlie this phenomenon. Calculations based on previously published results demonstrate that such a mechanism can explain the differences in the average duration of the interval encompassing a fast growth period and a slow growth period in in vitro microtubule assembly experiments in different conditions. Because no parameter unique to the microtubule assembly process is involved in the analysis, the proposed mechanism is expected to be generally applicable to heterogeneous chemical reactions. Also because biological systems are characterized by heterogeneous chemical reactions, the diffusion-based rhythmic characteristic of heterogeneous reactions is postulated to be a fundamental element in generating rhythmic behaviors in biological systems.

In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/Tardy Asynchronous Meiosis (TAM) gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele) frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products.